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WO2006062971A2 - Modulation des teneurs en carbone dans les plantes - Google Patents

Modulation des teneurs en carbone dans les plantes Download PDF

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Publication number
WO2006062971A2
WO2006062971A2 PCT/US2005/044112 US2005044112W WO2006062971A2 WO 2006062971 A2 WO2006062971 A2 WO 2006062971A2 US 2005044112 W US2005044112 W US 2005044112W WO 2006062971 A2 WO2006062971 A2 WO 2006062971A2
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WO
WIPO (PCT)
Prior art keywords
seq
nos
carbon
plant
promoter
Prior art date
Application number
PCT/US2005/044112
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English (en)
Other versions
WO2006062971A3 (fr
Inventor
Richard Schneeberger
Emilio Margolles-Clark
Joon-Hyun Park
Boris Jankowski
Steven Craig Bobzin
Original Assignee
Ceres Inc.
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Application filed by Ceres Inc. filed Critical Ceres Inc.
Publication of WO2006062971A2 publication Critical patent/WO2006062971A2/fr
Publication of WO2006062971A3 publication Critical patent/WO2006062971A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the material on the accompanying compact discs is hereby incorporated by reference into this application.
  • the accompanying compact discs contain one file, 60326849.txt, which was created on December 6, 2005.
  • the file named 60326849.txt is 386 kb.
  • the file can be accessed using Microsoft Word on a computer that uses Windows OS.
  • this document provides plants having increased carbon levels as well as materials and methods for making plants and plant products having increased carbon levels.
  • the ability of a plant to grow and develop under diverse and changing environmental conditions depends on the ability of the plant to utilize carbon and/or nitrogen. Specifically, the accumulation of one or both of these elements suggests that the plant is storing, synthesizing, or utilizing components such as nitrate, amino acids, proteins, sugars and/or carbohydrates to compensate for the changing environment.
  • the balance of carbon and nitrogen in plants is an important aspect of how plants utilize nitrogen efficiently. Carbon skeletons and energy are required in ample supply for nitrogen assimilation and re-assimilation (photorespiratory NH 4 ). Conversely, primary carbon assimilation is highly dependent on nitrogen assimilation because much of the nitrogen in a plant is invested in the proteins and chlorophyll of the photosynthetic machinery. Therefore, fixed carbon must be partitioned between amino acids and carbohydrate synthesis in a flexible manner that is responsive to the external and internal availability of nitrogen. There is a need for compositions and methods that can increase fixed carbon content under varying nitrogen conditions.
  • This document provides methods and materials related to plants having modulated (e.g., increased or decreased) levels of carbon.
  • this document provides transgenic plants and plant cells having increased levels of carbon, nucleic acids used to generate transgenic plants and plant cells having increased levels of carbon, and methods for making plants and plant cells having increased levels of carbon.
  • Such plants and plant cells can be grown to produce, for example, seeds having increased carbon content. Seeds having modulated carbon levels may be useful to produce foodstuffs and animal feed having increased or decreased oil, carbohydrate, and/or caloric content, which may benefit both food producers and consumers.
  • a method of modulating the level of carbon in a plant comprises introducing into a plant cell an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ ID NOs:67-81, SEQ ID NOs:83-92, and the consensus sequences set forth in Figures 1-7, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • a method of modulating the level of carbon in a plant comprises introducing into a plant cell an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and the consensus sequences set forth in Figures 1-7, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant
  • a method of modulating the level of carbon in a plant comprises introducing into a plant cell an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ TD NO:22, SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:40, SEQ E) NO:55, SEQ E) NO:57, SEQ TD NO:62, SEQ E) NO:67, SEQ E) NO:70, SEQ ID NO:72, SEQ TD NO:83, SEQ TD NO:85, SEQ TD NO:87, SEQ TD NO:88, SEQ E) NO:89, SEQ TD NO: 90, and SEQ TD NO:91, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of
  • the sequence identity can be 85 percent or greater, 90 percent or greater, or 95 percent or greater.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:2.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:22.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:36.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:40.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:55.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:67.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:83.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to a consensus sequence set forth in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, or Figure 7. The difference can be an increase in the level of carbon or oil.
  • the isolated nucleic acid can be operably linked to a regulatory region.
  • the regulatory region can be a tissue-specific regulatory region.
  • the tissue- specific regulatory region can be a promoter.
  • the promoter can be selected from the group consisting of YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter, the soybean trypsin inhibitor promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the soybean ⁇ ' subunit of ⁇ -conglycinin promoter, the oleosin promoter, the 15 kD zein promoter, the 16 kD zein promoter, the 19 kD zein promoter, the 22 IcD zein promoter, the 27 kD zein promoter, the Osgt-1 promoter, the beta-amylase gene promoter, and the barley hordein gene promoter.
  • the promoter can be selected from the group consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758.
  • the regulatory region can be a broadly expressing promoter.
  • the broadly expressing promoter can be selected from the group consisting of pl3879, p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633, YP0050, YP0144, and YP0190.
  • the regulatory region can be an inducible promoter.
  • the plant can be a dicot.
  • the plant can be a member of the genus Brassica, Glycine, Gossypium, Lactuca, Lycopersicon, Medicago, Solanum, Carthamus, Pisum, Trifolium, Helianthus, Arachis, Olea, Vitis, or Linum.
  • the plant can be a monocot.
  • the plant can be a member of the genus Zea, Triticum, Hordeum, Secale, Oryza, Triticosecale, Avena, Musa, Elaeis, Phleum, or Sorghum.
  • the tissue can be seed tissue.
  • a method of producing a plant tissue comprises growing a plant cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ ID NOs:67-81, SEQ ID NOs:83-92, and the consensus sequences set forth in Figures 1-7, where the tissue has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • a method of producing a plant tissue comprises growing a plant cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ TD NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and the consensus sequences set forth in Figures 1-7, where the tissue has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • a method of producing a plant tissue comprises growing a plant cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:83, SEQ TD NO:85, SEQ ID NO:87, SEQ E) NO:88, SEQ TD NO:89, SEQ E) NO: 90, and SEQ E) NO:91, where the tissue has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • the sequence identity can be 85 percent or greater, 90 percent or greater, or 95 percent or greater.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:2.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ E) NO:22.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ E)
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:40.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:55.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:67.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ TD NO:83.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to a consensus sequence set forth in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, or Figure 7. The difference can be an increase in the level of carbon or oil.
  • the isolated nucleic acid can be operably linked to a regulatory region.
  • the regulatory region can be a tissue-specific regulatory region.
  • the tissue- specific regulatory region can be a promoter.
  • the promoter can be selected from the group consisting of YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter, the soybean trypsin inhibitor promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the soybean ⁇ ' subunit of ⁇ -conglycinin promoter, the oleosin promoter, the 15 IdD zein promoter, the 16 kD zein promoter, the 19 kD zein promoter, the 22 IdD zein promoter, the 27 IdD zein promoter, the Osgt-1 promoter, the beta- amylase gene promoter, and the barley hordein gene promoter.
  • the promoter can be selected from the group consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758.
  • the regulatory region can be a broadly expressing promoter.
  • the broadly expressing promoter can be selected from the group consisting of pl3879, p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633, YP0050, YP0144, and YP0190.
  • the regulatory region can be an inducible promoter.
  • the plant tissue can be dicotyledonous.
  • the plant tissue can be a member of the genus Brassica, Glycine, Gossypium, Lactuca, Lycopersicon, Medicago, Solanum, Carthamus, Pisum, Trifolium, Helianthus, Arachis, Olea, Vitis, ovLinum.
  • the plant tissue can be monocotyledonous.
  • the plant tissue can be a member of the genus Zea, Triticum, Hordeum, Secale, Oryza, Triticosecale, Avena, Musa, Elaeis, Phleum, or Sorghum.
  • the tissue can be seed tissue.
  • a plant cell comprises an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ BD NOs:67-81, SEQ ID NOs:83-92, and the consensus sequences set forth in Figures 1 -7, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • a plant cell comprises an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, SEQ TD NO:67, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and the consensus sequences set forth in Figures 1 -7, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • a plant cell comprises an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO.37, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:91, where a tissue of a plant produced from the plant cell has a difference in the level of carbon as compared to the corresponding level in tissue of a control plant that does not comprise the nucleic acid.
  • the sequence identity can be 85 percent or greater, 90 percent or greater, or 95 percent or greater.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:2.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:22.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:36.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:40.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:55.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO: 67.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:83.
  • the nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding to a consensus sequence set forth in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, or Figure 7. The difference can be an increase in the level of carbon or oil.
  • the isolated nucleic acid can be operably linked to a regulatory region.
  • the regulatory region can be a tissue-specific regulatory region.
  • the tissue- specific regulatory region can be a promoter.
  • the promoter can be selected from the group consisting of YP0092, PT0676, PT0708, the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter, the soybean trypsin inhibitor promoter, the ACP promoter, the stearoyl-ACP desaturase gene, the soybean ⁇ ' subunit of ⁇ -conglycinin promoter, the oleosin promoter, the 15 kD zein promoter, the 16 kD zein promoter, the 19 IcD zein promoter, the 22 kD zein promoter, the 27 IdD zein promoter, the Osgt-1 promoter, the beta- amylase gene promoter, and the barley hordein gene promoter.
  • the promoter can be selected from the group consisting of PT0613, PT0672, PT0678, PT0688, PT0837, YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758.
  • the regulatory region can be a broadly expressing promoter.
  • the broadly expressing promoter can be selected from the group consisting of pl3879, p32449, 21876, p326, YP0158, YP0214, YP0380, PT0848, PT0633, YP0050, YP0144, and YP0190.
  • the regulatory region can be an inducible promoter.
  • the plant can be a dicot.
  • the plant can be a member of the genus Brassica, Glycine, Gossypium, Lactuca, Lycopersicon, Medicago, Solarium, Carthamus, Pisum, Trifolium, Helianthus, Arachis, Olea, Vitis, or Linum.
  • the plant can be a monocot.
  • the plant can be a member of the genus Zea, Triticum, Hordeum, Secale, Oryza, Triticosecale, Avena, Musa, Elaeis, Phleum, or Sorghum.
  • the tissue can be seed tissue.
  • a transgenic plant is also provided.
  • the transgenic plant comprises any of the plant cells described above. Progeny of the transgenic plant are also provided.
  • the progeny have a difference in the level of carbon as compared to the level of carbon in a corresponding control plant that does not comprise the isolated nucleic acid.
  • Seed and vegetative tissue from the transgenic plant are also provided.
  • food products and feed products comprising vegetative tissue from the transgenic plant are provided.
  • Figure 1 is an alignment of SEQ ID NO:2 with orthologous amino acid sequences SEQ ID NOs:3-7, SEQ ID NOs:10-ll, SEQ ID NOs:13-15, and SEQ ID NO: 17. The consensus sequence determined by the alignment is set forth.
  • Figure 2 is an alignment of SEQ ID NO:22 with orthologous amino acid sequences SEQ ID NOs:23-27, SEQ ID NO:32, and SEQ TD NO:33. The consensus sequence determined by the alignment is set forth.
  • Figure 3 is an alignment of SEQ ID NO:36 with homologous and orthologous amino acid sequences SEQ ID NOs:37-38. The consensus sequence determined by the alignment is set forth.
  • Figure 4 is an alignment of SEQ ID NO:40 with orthologous amino acid sequences SEQ ID NO:41, SEQ ID NO:48, and SEQ ID NOs:51-53. The consensus sequence determined by the alignment is set forth.
  • Figure 5 is an alignment of SEQ ID NO:55 with homologous and orthologous amino acid sequences SEQ ID NOs:61-65. The consensus sequence determined by the alignment is set forth.
  • Figure 6 is an alignment of SEQ ID NO:67 with homologous and orthologous amino acid sequences SEQ ID NOs:71-75, SEQ ID NOs:77-78, and SEQ ID NO:81. The consensus sequence determined by the alignment is set forth.
  • Figure 7 is an alignment of SEQ ID NO:83 with orthologous amino acid sequences SEQ ID NOs:87-88 and SEQ ID NOs:91-92. The consensus sequence determined by the alignment is set forth. DETAILED DESCRIPTION
  • the invention features methods and materials related to modulating (e.g., increasing or decreasing) carbon levels in plants.
  • the plants may also have modulated levels of nitrogen.
  • the methods can include transforming a plant cell with a nucleic acid encoding a carbon-modulating polypeptide, wherein expression of the polypeptide results in a modulated level of carbon.
  • Plant cells produced using such methods can be grown to produce plants having an increased or decreased carbon content. Seeds from such plants may be used to produce, for example, foodstuffs and animal feed having increased or decreased oil, carbohydrate, and/or caloric content, which may benefit both food producers and consumers.
  • polypeptide refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation.
  • the subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including D/L optical isomers. Full- length proteins, analogs, mutants, and fragments thereof are encompassed by this definition.
  • Carbon-modulating polypeptides can be effective to modulate carbon levels when expressed in a plant or plant cell. Modulation of the level of carbon can be either an increase or a decrease in the level of carbon relative to the corresponding level in a control plant.
  • a carbon-modulating polypeptide can be a transporter polypeptide, such as a nitrate, proline, ammonium, or oligopeptide transporter polypeptide.
  • a carbon-modulating polypeptide can also be an enzyme, such as anthranilate synthase, that catalyzes a reaction in an amino acid biosynthetic pathway.
  • a carbon-modulating polypeptide can be a nitrate transporter polypeptide, such as a NRT2.5 nitrate transporter polypeptide.
  • Nitrate transporter polypeptides are involved in the nitrate uptake process in plants.
  • SEQ ID NO:2 sets forth the amino acid sequence of an Arahidopsis clone, identified herein as Ceres cDNA ID 3080447 (SEQ ID NO:1), that is predicted to encode a NRT2.5 nitrate transporter polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:2.
  • a carbon-modulating polypeptide can be a homo log, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 55% sequence identity, e.g., 56%, 57%, 58%, 59%, 60%, 61%, 65%, 66%, 67%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:2.
  • Amino acid sequences of orthologs of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 are provided in Figure 1, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:2, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 1 provides the amino acid sequences of Ceres cDNA TD 3080447 (SEQ ID NO:2), gi
  • orthologs include gi
  • a carbon-modulating polypeptide includes a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ E) NO:2, SEQ TD NO:3, SEQ TD NO:4, SEQ TD NO:5, SEQ E) NO:6, SEQ TD NO:7, SEQ E) NO:8, SEQ TD NO:9, SEQ TD NO:10, SEQ TD NO:11, SEQ E ) NO: 12, SEQ E ) NO:13, SEQ E ) NO:14, SEQ E) NO:15, SEQ TD NO:16, SEQ E ) NO: 17, SEQ TD NO:18, SEQ TD NO:19, SEQ TD NO:20, or the consensus sequence set forth in Figure 1.
  • a carbon-modulating polypeptide can be an ammonium transporter polypeptide, such as an ATMl ;2 ammonium transporter polypeptide.
  • Ammonium transporter polypeptides are involved in regulating ammonium uptake in plants.
  • SEQ ID NO:22 sets forth the amino acid sequence of an Ar ⁇ bidopsis clone, identified herein as Ceres cDNA ID 1828694 (SEQ ID NO:21), that is predicted to encode an ATM1;2 ammonium transporter polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:22.
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ TD NO:22.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 65% sequence identity, e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 76%, 77%, 79%, 80%, 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:22.
  • Amino acid sequences of orthologs of the polypeptide having the amino acid sequence set forth in SEQ TD NO:22 are provided in Figure 2, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:22, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 2 provides the amino acid sequences of Ceres cDNA ID 1828694 (SEQ ID NO:22), gi
  • orthologs include gi
  • a carbon-modulating polypeptide includes a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or the consensus sequence set forth in Figure 2.
  • a carbon-modulating polypeptide can be a proton-dependent oligopeptide transport (POT) family polypeptide.
  • POT family polypeptides seem to be mainly involved in the intake of small peptides with the concomitant uptake of a proton.
  • SEQ ID NO:36 sets forth the amino acid sequence of an Arabidopsis clone, identified herein as Ceres cDNA ID 3086062 (SEQ ID NO:35), that has a PTR2 domain characteristic of a peptide transporter polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:36.
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:36.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 45% sequence identity, e.g., 46%, 47%, 48%, 49%, 50%, 51%, 55%, 60%, 61%, 63%, 63%, 66%, 61%, 68%, 69%, 70%, 71 %, 72%, 75%, 76%, 77%, 79%, 80%, 81 %, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:36.
  • Amino acid sequences of a homolog and an ortholog of the polypeptide having the amino acid sequence set forth in SEQ ID NO:36 are provided in Figure 3, along with a consensus sequence.
  • a consensus amino acid sequence for such a homolog and ortholog was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:36, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 3 provides the amino acid sequences of Ceres cDNA ID 3086062 (SEQ ID NO:36), CeresClone: 1002997 (SEQ ID NO:37), and gi
  • a carbon-modulating polypeptide can include a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or the consensus sequence set forth in Figure 3.
  • a carbon-modulating polypeptide can also comprise the amino acid sequence of an Arabidopsis clone, identified herein as Ceres cDNA ID 3091277 SEQ ID NO:39), that is predicted to encode a POT family polypeptide (SEQ ID NO:40).
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:40.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 35% sequence identity, e.g., 36%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 55%, 60%, 61%, 63%, 63%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 76%, 77%, 79%, 80%, 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:40.
  • Amino acid sequences of orthologs of the polypeptide having the amino acid sequence set forth in SEQ TD NO:40 are provided in Figure 4, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:40, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 4 provides the amino acid sequences of Ceres cDNA ID 3091277 (SEQ ID NO:40), gi
  • Other orthologs include gi
  • a carbon-modulating polypeptide can include a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or the consensus sequence set forth in Figure 4.
  • a carbon-modulating polypeptide can be a transporter polypeptide.
  • Transporter polypeptides allow uptake of essential nutrients and ions, excretion of end products of metabolism and deleterious substances, and communication between cells and the environment. Transporter polypeptides also provide essential constituents of energy-generating and energy-consuming systems.
  • SEQ ID NO:55 sets forth the amino acid sequence of an Arabidopsis clone, identified herein as Ceres cDNA ID 2997404 (SEQ ID NO:54), that has a PTR2 domain characteristic of a transporter polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:55.
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:55.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 30% sequence identity, e.g., 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 55%, 60%, 61%, 63%, 63%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 75%, 76%, 77%, 79%, 80%, 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:55.
  • Amino acid sequences of orthologs and a homolog of the polypeptide having the amino acid sequence set forth in SEQ ID NO:55 are provided in Figure 5, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs and homolog was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:55, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 5 provides the amino acid sequences of Ceres cDNA ID 2997404 (SEQ ID NO:55), gi
  • orthologs and homologs include gi
  • a carbon-modulating polypeptide can include a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or the consensus sequence set forth in Figure 5.
  • a carbon-modulating polypeptide can be a proline transporter polypeptide, such as a ProTl proline transporter polypeptide.
  • Proline transporter polypeptides transport proline but not other amino acids in plants.
  • SEQ ID NO:67 sets forth the amino acid sequence of an Arabidopsis clone, identified herein as Ceres cDNA ID 4904707 (SEQ ID NO: 66), that is predicted to encode a proline transporter polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:67.
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO:67.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 60% sequence identity, e.g., 61%, 65%, 66%, 67%, 69%, 70%, 71%, 74%, 75%, 80%, 85%, 90%, 91%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 67.
  • Amino acid sequences of orthologs and a homolog of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 67 are provided in Figure 6, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs and homolog was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:67, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 6 provides the amino acid sequences of Ceres cDNA ID 4904707 (SEQ ID NO:67), gi
  • homologs and orthologs include gi
  • a carbon-modulating polypeptide can include a polypeptide having at least 80% sequence identity, e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid sequence corresponding to SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ E) NO:76, SEQ E) NO:77, SEQ ID NO:78, SEQ E ) NO:79,
  • a carbon-modulating polypeptide can be a subunit of an enzyme, such as anthranilate synthase, that catalyzes a reaction in an amino acid biosynthetic pathway.
  • Anthranilate synthase catalyzes the first reaction in the tryptophan biosynthetic pathway.
  • SEQ ID NO: 83 sets forth the amino acid sequence of an Arabidopsis clone, identified herein as Ceres cDNA ID 5669462 (SEQ TD NO:82), that is predicted to encode an anthranilate synthase beta chain polypeptide.
  • a carbon-modulating polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:83.
  • a carbon-modulating polypeptide can be a homolog, ortholog, or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 83.
  • a carbon-modulating polypeptide can have an amino acid sequence with at least 65% sequence identity, e.g., 66%, 67%, 69%, 70%, 71%, 74%, 75%, 80%, 85%, 90%, 91%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:83.
  • Amino acid sequences of orthologs of the polypeptide having the amino acid sequence set forth in SEQ ID NO:83 are provided in Figure 7, along with a consensus sequence.
  • a consensus amino acid sequence for such orthologs was determined by aligning amino acid sequences, e.g., amino acid sequences related to SEQ ID NO:83, from a variety of species and determining the most common amino acid or type of amino acid at each position.
  • the alignment in Figure 7 provides the amino acid sequences of Ceres cDNA ID 5669462 (SEQ ID NO:83), CeresClone:967151 (SEQ ID NO:87), CeresClone:214246 (SEQ ID NO:88), CeresClone:686561 (SEQ ID NO:91), and gi
  • orthologs and homologs include gi
  • a carbon-modulating polypeptide can include a polypeptide having at least 80% sequence identity (e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity) to an amino acid sequence corresponding to SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or the consensus sequence set forth in Figure 7.
  • sequence identity e.g., 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity
  • nucleic acids can encode a polypeptide having a particular amino acid sequence.
  • the degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given carbon-modulating polypeptide can be modified such that optimal expression in a particular plant species is obtained, using appropriate codon bias tables for that species.
  • a carbon-modulating polypeptide encoded by a recombinant nucleic acid can be a native carbon-modulating polypeptide, i.e., one or more additional copies of the coding sequence for a carbon-modulating polypeptide that is naturally present in the cell.
  • a carbon-modulating polypeptide can be heterologous to the cell, e.g., a transgenic Lycopersicon plant can contain the coding sequence for a transporter polypeptide from a Glycine plant.
  • a carbon-modulating polypeptide can include additional amino acids that are not involved in carbon modulation, and thus can be longer than would otherwise be the case.
  • a carbon-modulating polypeptide can include an amino acid sequence that functions as a reporter.
  • Such a carbon- modulating polypeptide can be a fusion protein in which a green fluorescent protein (GFP) polypeptide is fused to SEQ ID NO:2, or in which a yellow fluorescent protein (YFP) polypeptide is fused to SEQ E) NO:22.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • a carbon-modulating polypeptide includes a purification tag or a leader sequence added to the amino or carboxy terminus.
  • Carbon-modulating polypeptide candidates suitable for use in the invention can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify orthologs of carbon-modulating polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using known carbon- modulating polypeptide amino acid sequences. Those proteins in the database that have greater than 40% sequence identity can be identified as candidates for further evaluation for suitability as a carbon-modulating polypeptide. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains suspected of being present in carbon-modulating polypeptides, e.g., conserved functional domains.
  • conserved regions in a template or subject polypeptide can facilitate production of variants of wild type carbon-modulating polypeptides.
  • conserved regions can be identified by locating a region within the primary amino acid sequence of a template polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains at sanger.ac.uk/Pfam and genome.wustl.edu/Pfam. A description of the information included at the Pfam database is described in Sonnhammer et ah, 1998, Nucl. Acids Res. 26: 320-322; Sonnhammer et ah, 1997, Proteins 28:405-420; and Bateman et ah, 1999, Nucl. Adds Res. 27:260-262.
  • conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate. For example, sequences from Arabidopsis and Zea mays can be used to identify one or more conserved regions.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides can exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity), hi some embodiments, a conserved region of target and template polypeptides exhibit at least 92, 94, 96, 98, or 99% amino acid sequence identity.
  • Amino acid sequence identity can be deduced from amino acid or nucleotide sequences. In certain cases, highly conserved domains have been identified within carbon-modulating polypeptides. These conserved regions can be useful in identifying functionally similar (orthologous) carbon- modulating polypeptides.
  • suitable carbon-modulating polypeptides can be synthesized on the basis of consensus functional domains and/or conserved regions in polypeptides that are homologous carbon-modulating polypeptides.
  • Domains are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint” or "signature” that can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities.
  • a domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
  • Consensus domains and conserved regions can be identified by homologous polypeptide sequence analysis as described above. The suitability of polypeptides for use as carbon-modulating polypeptides can be evaluated by functional complementation studies.
  • nucleic acid and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs. Polynucleotides can have any three- dimensional structure. A nucleic acid can be double-stranded or single-stranded ⁇ i.e., a sense strand or an antisense strand).
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA siRNA
  • micro-RNA micro-RNA
  • ribozymes cDNA
  • recombinant polynucleotides branched polynucleotides
  • plasmids vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
  • An isolated nucleic acid can be, for example, a naturally-occurring DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences ⁇ e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment).
  • An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR polymerase chain reaction
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule ⁇ e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
  • percent sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
  • a subject sequence typically has a length that is more than 80 percent, e.g., more than 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120 percent, of the length of the query sequence.
  • a query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). Chenna, et al. (2003) Nucleic Acids Res 31 (13):3497-500.
  • ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • word size 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3.
  • weight matrix blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: GIy, Pro, Ser, Asn, Asp, GIn, GIu, Arg, and Lys; residue-specific gap penalties: on.
  • the output is a sequence alignment that reflects the relationship between sequences.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi . ac .uk/clustalw) .
  • ClustalW divides the number of identities in the best alignment by the number of residues compared (gap positions are excluded), and multiplies the result by 100.
  • the output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • exogenous nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism.
  • exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration.
  • a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant.
  • a recombinant nucleic acid construct comprises a nucleic acid encoding a carbon- modulating polypeptide as described herein, operably linked to a regulatory region suitable for expressing the carbon-modulating polypeptide in the plant or cell.
  • a nucleic acid can comprise a coding sequence that encodes any of the carbon-modulating polypeptides as set forth in SEQ E) NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ ID NOs:67-81, SEQ ID NOs:83-92, and the consensus sequences set forth in Figures 1-7.
  • Vectors containing nucleic acids such as those described herein also are provided.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An “expression vector” is a vector that includes a regulatory region.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., chlorosulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FlagTM tag FlagTM tag
  • regulatory region refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • operably linked refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically comprises at least a core (basal) promoter.
  • a promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a suitable enhancer is a cis-regulatory element (-212 to - 154) from the upstream region of the octopine synthase (ocs) gene. Fromm et ah, The Plant Cell 1 :977-984 (1989).
  • the choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue- preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.
  • a promoter that is active predominantly in a reproductive tissue e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, zygote, endosperm, integument, or seed coat
  • a reproductive tissue e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, zygote, endosperm, integument, or seed coat
  • a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well.
  • Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et ah, Plant Cell, 1:855- 866 (1989); Bustos, et ah, Plant Cell, 1:839-854 (1989); Green, et ah, EMBO J. 7, 4035-4044 (1988); Meier, et ah, Plant Cell, 3, 309-316 (1991); and Zhang, et ah, Plant Physiology 110: 1069-1079 (1996). Examples of various classes of promoters are described below. Some of the promoters indicated below are described in more detail in U.S. Patent Application Ser. Nos.
  • a promoter may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species. Nucleotide sequences of promoters are set forth in SEQ ID NOs:93-100. Broadly Expressing; Promoters
  • a promoter can be said to be "broadly expressing" when it promotes transcription in many, but not necessarily all, plant tissues.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds.
  • Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO:94), YP0144 (SEQ ID NO:95), YP0190 (SEQ ID NO:96), pl3879 (SEQ ID NO:97), YP0050 (SEQ ID NO:98), p32449 (SEQ ID NO:99), 21876 (SEQ ID NO: 100), YP0158, YP0214, YP0380, PT0848, and PT0633 promoters.
  • CaMV 35S promoter the cauliflower mosaic virus (CaMV) 35S promoter
  • MAS mannopine synthase
  • 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens the figwort mosaic virus 34S promoter
  • actin promoters such as the rice actin promoter
  • ubiquitin promoters such as the maize ubiquitin-1 promoter.
  • the CaMV 35S promoter is excluded from the category of broadly expressing promoters.
  • Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues.
  • root-active promoters are root-preferential promoters, i.e., confer transcription only or predominantly in root tissue.
  • Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758 promoters.
  • Other root-preferential promoters include the PT0613, PT0672, PT0688, and PT0837 promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds.
  • root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et ai, Proc. Natl. Acad. ScL USA 86:7890-7894 (1989)), root cell specific promoters reported by Conkling et al, Plant Physiol. 93:1203-1211 (1990), and the tobacco RD2 gene promoter.
  • promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used.
  • Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter (Bustos et al, Plant Cell l(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs et al, Plant Cell l(6):609-621 (1989)), the ACP promoter (Baerson et al, Plant MoI Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase gene (Slocombe et al, Plant Physiol 104(4): 167- 176 (1994)), the soybean ⁇ ' subunit of ⁇ -conglycinin promoter (Chen et al, Proc Natl Acad Sd USA 83:8560-8564 (1986)), the oleosin promoter (Hong et al, Plant MoI Biol 34(3):549
  • Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al, MoI Cell Biol. 13:5829-5842 (1993)), the beta-amylase gene promoter, and the barley hordein gene promoter.
  • Other maturing endosperm promoters include the YP0092, PT0676, and PT0708 promoters.
  • Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, and the melon actin promoter.
  • promoters that are active primarily in ovules include YP0007, YPOl 11, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YPOl 15, YPOl 19, YP0120, and YP0374.
  • Embryo Sac/Early Endosperm Promoters To achieve expression in embryo sac/early endosperm, regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
  • Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoI. Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent
  • promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant MoI. Biol, 22:10131-1038).
  • Other promoters include the following Arabidopsis promoters: YP0039, YPOlOl, YP0102, YPOl 10, YPOl 17, YPOl 19, YP0137, DME,
  • promoters that may be useful include the following rice promoters: ⁇ 530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285.
  • Embryo Promoters Regulatory regions that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable. Embryo-preferential promoters include the barley lipid transfer protein (Ltpl) promoter ⁇ Plant Cell Rep (2001) 20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, and PT0740.
  • Ltpl barley lipid transfer protein
  • Photosynthetic Tissue Promoters Active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricind), the pine cab6 promoter (Yamamoto et al, Plant Cell Physiol. 35:773-778 (1994)), the Cab-1 gene promoter from wheat (Fejes et al, Plant MoI. Biol.
  • RbcS ribulose-l,5-bisphosphate carboxylase
  • Inducible Promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli.
  • inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought.
  • drought- inducible promoters include YP0380, PT0848, YP0381, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PD1367, PD0901, and PD0898.
  • Nitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.
  • An example of a shade-inducible promoter is PR0924.
  • Basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation.
  • Basal promoters frequently include a "TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation.
  • Basal promoters also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
  • promoters include, but are not limited to, leaf- preferential, stem/shoot-preferential, callus-preferential, guard cell-preferential, such as PT0678, and senescence-preferential promoters. Promoters designated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YPOl 19, and YP0096, as described in the above-referenced patent applications, may also be useful.
  • a 5' untranslated region can be included in nucleic acid constructs described herein.
  • a 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide.
  • a 3' UTR can be positioned between the translation termination codon and the end of the transcript.
  • UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
  • more than one regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • more than one regulatory region can be operably linked to the sequence of a polynucleotide encoding a carbon-modulating polypeptide.
  • Transgenic Plants and Plant Cells The invention also features transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein.
  • a plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division.
  • a plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
  • Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. Progeny include descendants of a particular plant or plant line.
  • Progeny of an instant plant include seeds formed on F 1 , F 2 , F 3 , F 4 , F 5 , F 6 and subsequent generation plants, or seeds formed on BC 1 , BC 2 , BC 3 , and subsequent generation plants, or seeds formed on F 1 BC 1 , F 1 BC 2 , F 1 BC 3 , and subsequent generation plants.
  • the designation Fi refers to the progeny of a cross between two parents that are genetically distinct.
  • the designations F 2 , F 3 , F 4 , F 5 and F 6 refer to subsequent generations of self- or sib-pollinated progeny of an Fi plant. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.
  • Transgenic plants can be grown in suspension culture, or tissue or organ culture.
  • solid and/or liquid tissue culture techniques can be used.
  • transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium.
  • transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium.
  • Solid medium typically is made from liquid medium by adding agar.
  • a solid medium can be Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4- dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin.
  • an auxin e.g., 2,4- dichlorophenoxyacetic acid (2,4-D)
  • a cytokinin e.g., kinetin.
  • a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation.
  • a suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days.
  • transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous carbon-modulating polypeptide whose expression has not previously been confirmed in particular recipient cells.
  • Techniques for introducing nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mQdiatQd transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including dicots such as alfalfa, amaranth, apple, beans (including kidney beans, lima beans, dry beans, green beans), broccoli, cabbage, carrot, castor bean, cherry, chick peas, chicory, clover, cocoa, coffee, cotton, crambe, flax, grape, grapefruit, lemon, lentils, lettuce, linseed, mango, melon (e.g., watermelon, cantaloupe), mustard, orange, peach, peanut, pear, peas, pepper, plum, potato, oilseed rape, rapeseed (high erucic acid and canola), safflower, sesame, soybean, spinach, strawberry, sugar beet, sunflower, sweet potatoes, tea, tomato, and yams, as well as monocots such as banana, barley, bluegrass,
  • the methods and compositions described herein can be used with dicotyledonous plants belonging, for example, to the orders Apiales, Arecales, Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Cucurbitales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Linales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papaverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranun
  • compositions described herein also can be utilized with monocotyledonous plants such as those belonging to the orders Alismatales, Arales, Arecales, Asparagales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales,
  • Gymnospermae plants belonging to Gymnospermae, e.g., Cycadales, Ginkgoales, Gnetales, and Pinales.
  • compositions can be used over a broad range of plant species, including species from the dicot genera Amaranthus, Arachis, Brassica, Calendula, Camellia, Capsicum, Carthamus, Cicer, Cichorium, Cinnamomum, Citrus, Citrullus, Coffea, Crambe, Cucumis, Cucurbita, Daucus, Dioscorea, Fragaria, Glycine, Gossypium, Helianthus, Lactuca, Lens, Linum, Lycopersicon, Malus, Mangifera, Medicago, Mentha, Nicotiana, Ocimum, Olea, Phaseolus, Pistacia, Pisum, Prunus, Pyrus, Rosmarinus, Salvia, Sesamum, Solatium, Spinacia, Theobroma, Thymus, Trifolium, Vaccinium, Vigna, and Vitis; and the monocot genera Allium, Ananas, Asparagus, Avena, Curcuma
  • brown seaweeds e.g., Ascophyllum nodosum, Fucus vesiculosus, Fucus serratus, Himanthalia elongata, and Undaria pinnatifida
  • red seaweeds e.g., Chondrus crispus, Cracilaria verrucosa, Porphyra umbilicalis, and Palmaria palmata
  • green seaweeds e.g., Enteromorpha spp. and Ulva spp.
  • microalgae e.g., Spirulina spp. (S. platensis and S. maxima) and Odontella aurita.
  • a plant is a member of the species Ananus comosus, Brassica campestris, Brassica napus, Brassica oleracea, Glycine max, Gossypium spp., Lactuca sativa, Lycopersicon esculentum, Musa paradisiaca, Oryza sativa, Solanum tuberosum, Triticum aestivum, Vitis vinifera, or Zea mays.
  • the polynucleotides and recombinant vectors described herein can be used to express or inhibit expression of a carbon-modulating polypeptide in a plant species of interest.
  • expression refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes.
  • Up-regulation” or “activation” refers to regulation that increases the production of expression products (mRNA, polypeptide, or both) relative to basal or native states
  • down-regulation or “repression” refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states.
  • nucleic-acid based methods including anti-sense RNA, ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be used to inhibit protein expression in plants.
  • Antisense technology is one well-known method. In this method, a nucleic acid segment from the endogenous gene is cloned and operably linked to a promoter so that the antisense strand of RNA is transcribed. The recombinant vector is then transformed into plants, as described above, and the antisense strand of RNA is produced.
  • the nucleic acid segment need not be the entire sequence of the endogenous gene to be repressed, but typically will be substantially identical to at least a portion of the endogenous gene to be repressed.
  • a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
  • an isolated nucleic acid provided herein can be an antisense nucleic acid to one of the aforementioned nucleic acids encoding a carbon-modulating polypeptide, e.g., SEQ ID NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ ID NOs:67-81, SEQ ID NOs:83-92, or a consensus sequence set forth in Figures 1-7.
  • a carbon-modulating polypeptide e.g., SEQ ID NOs:2-20, SEQ ID NOs:22-34, SEQ ID NOs:36-38, SEQ ID NOs:40-43, SEQ ID NO:48, SEQ ID NOs:51-53, SEQ ID NOs:55-65, SEQ ID NOs:67-81, SEQ ID NOs:83-92, or a
  • a nucleic acid that decreases the level of a transcription or translation product of a gene encoding a carbon-modulating polypeptide is transcribed into an antisense nucleic acid similar or identical to the sense coding sequence of the carbon-modulating polypeptide.
  • the transcription product of an isolated nucleic acid can be similar or identical to the sense coding sequence of a carbon-modulating polypeptide, but is an RNA that is unpolyadenylated, lacks a 5' cap structure, or contains an unsplicable intron.
  • a nucleic acid in another method, can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
  • Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
  • Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • RNA contains a 5'-UG-3' nucleotide sequence.
  • the construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein.
  • Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
  • tRNA transfer RNA
  • RNA endoribonucleases such as the one that occurs naturally in Tetrahymena thermophila, and which have been described extensively by Cech and collaborators can be useful. See, for example, U.S. Patent No. 4,987,071.
  • RNA interference is a cellular mechanism to regulate the expression of genes and the replication of viruses. This mechanism is thought to be mediated by double- stranded small interfering RNA molecules. A cell responds to such a double- stranded RNA by destroying endogenous mRNA having the same sequence as the double-stranded RNA.
  • Methods for designing and preparing interfering RNAs are known to those of skill in the art; see, e.g., WO 99/32619 and WO 01/75164. For example, a construct can be prepared that includes a sequence that is transcribed into an interfering RNA.
  • Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure.
  • One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of the polypeptide of interest, and that is from about 10 nucleotides to about 2,500 nucleotides in length.
  • the length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides.
  • the other strand of the stem portion of a double stranded RNA comprises an antisense sequence of the carbon-modulating polypeptide of interest, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence.
  • the loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides.
  • the loop portion of the RNA can include an intron. See, e.g., WO 99/53050.
  • nucleic-acid based methods for inhibition of gene expression in plants can be a nucleic acid analog.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Sumrnerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et ah, 1996, Bioorgan. Med. Chem., 4: 5-23.
  • the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • a transformed cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered plant material for particular traits or activities, e.g., those encoded by marker genes or antibiotic resistance genes. Such screening and selection methodologies are well known to those having ordinary skill in the art. In addition, physical and biochemical methods can be used to identify transformants.
  • Transgenic plants can have an altered phenotype as compared to a corresponding control plant that either lacks the transgene or does not express the transgene.
  • a polypeptide can affect the phenotype of a plant ⁇ e.g., a transgenic plant) when expressed in the plant, e.g., at the appropriate time(s), in the appropriate tissue(s), or at the appropriate expression levels.
  • Phenotypic effects can be evaluated relative to a control plant that does not express the exogenous polynucleotide of interest, such as a corresponding wild type plant, a corresponding plant that is not transgenic for the exogenous polynucleotide of interest but otherwise is of the same genetic background as the transgenic plant of interest, or a corresponding plant of the same genetic background in which expression of the polypeptide is suppressed, inhibited, or not induced ⁇ e.g., where expression is under the control of an inducible promoter).
  • a plant can be said "not to express" a polypeptide when the plant exhibits less than 10%, e.g.
  • a polypeptide or mRNA encoding the polypeptide exhibited by the plant of interest Expression can be evaluated using methods including, for example, RT-PCR, Northern blots, Sl RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays, and mass spectrometry. It should be noted that if a polypeptide is expressed under the control of a tissue-specific or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polypeptide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period.
  • a plant in which expression of a carbon- modulating polypeptide is modulated can have increased levels of seed carbon.
  • a carbon-modulating polypeptide described herein can be expressed in a transgenic plant, resulting in increased levels of seed carbon.
  • the seed carbon level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or more than 45 percent, as compared to the seed carbon level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a carbon-modulating polypeptide is modulated can have decreased levels of seed carbon.
  • the seed carbon level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the seed carbon level in a corresponding control plant that does not express the transgene.
  • Plants for which modulation of levels of seed carbon can be useful include, without limitation, alfalfa, lettuce, carrot, onion, broccoli, tomato, potato, sugarcane, grape, cotton, canola, sweet corn, popcorn, field corn, peas, beans, safflower, soybean, coffee, amaranth, rapeseed, peanut, sunflower, oil palm, corn, clover, wheat, rye, barley, oat, rice, millet, strawberry, pineapple, melon, peach, pear, apple, cherry, orange, lemon, grapefruit, plum, mango, banana, clover, sudangrass, switchgrass, and sorghum.
  • a plant in which expression of a carbon- modulating polypeptide is modulated can have increased or decreased levels of fixed carbon in one or more non-seed tissues, e.g., leaf tissues, stem tissues, root or corm tissues, or fruit tissues other than seed.
  • the carbon level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or more than 45 percent, as compared to the carbon level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a carbon-modulating polypeptide is modulated can have decreased levels of fixed carbon in one or more non-seed tissues.
  • the carbon level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the carbon level in a corresponding control plant that does not express the transgene.
  • Plants for which modulation of levels of fixed carbon in non-seed tissues can be useful include, without limitation, alfalfa, lettuce, carrot, onion, broccoli, tomato, potato, peanut, sugarcane, sudangrass, grape, timothy, strawberry, pineapple, melon, peach, pear, apple, cherry, orange, lemon, grapefruit, plum, mango, banana, grand fir, balsam fir, yellow pine, jack pine, loblolly pine, white pine, blue spruce, poplar, fescue, ryegrass, bluegrass and switchgrass.
  • Increases in non-seed carbon in such plants can provide improved renewable energy sources; increased oil, carbohydrate, and/or caloric content in edible plants; increased production of building materials; or increased production of animal forage.
  • a plant in which expression of a carbon- modulating polypeptide having an amino acid sequence corresponding to SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:55, or SEQ ID NO:67 is modulated can have decreased levels of seed nitrogen accompanying increased levels of seed carbon.
  • the nitrogen level can be decreased by at least 5 percent, . e.g., 5, 10, 15, 20, 25, 30, 35, or 40 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a carbon- modulating polypeptide having an amino acid sequence corresponding to SEQ ID NO:83 is modulated can have an increased seed nitrogen level accompanying an increased seed carbon level.
  • the nitrogen level can be increased by at least 5 percent, e.g., 5, 10, 15, 20, 25, or 30 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a difference e.g., an increase
  • a difference in the amount of carbon or nitrogen in a transgenic plant or cell relative to a control plant or cell is considered statistically significant at p ⁇ 0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t-test, Mann- Whitney test, or F-test.
  • a difference in the amount of carbon or nitrogen is statistically significant at p ⁇ 0.01, p ⁇ 0.005, or p ⁇ 0.001.
  • a statistically significant difference in, for example, the amount of carbon in a transgenic plant compared to the amount in cells of a control plant indicates that (1) the recombinant nucleic acid present in the transgenic plant results in altered carbon levels and/or (2) the recombinant nucleic acid warrants further study as a candidate for altering the amount of carbon in a plant.
  • articles of manufacture that comprise seeds from transgenic plants provided herein.
  • the seeds can be conditioned using means known in the art and packaged using packaging material well known in the art to prepare an article of manufacture.
  • a package of seed can have a label e.g., a tag or label secured to the packaging material, a label printed on the packaging material or a label inserted within the package.
  • the label can indicate that plants grown from the seeds contained within the package can produce a crop having an altered level of carbon relative to corresponding control plants.
  • Example 1 Transgenic plants The following symbols are used in the Examples: T 1 : first generation transformant; T 2 : second generation, progeny of self-pollinated T 1 plants; T 3 : third generation, progeny of self-pollinated T 2 plants; T 4 : fourth generation, progeny of self-pollinated T 3 plants. Independent transformations are referred to as events.
  • Ceres cDNA ID 3080447 (SEQ ID NO:1) is a cDNA clone that is predicted to encode a 502 amino acid (SEQ ID NO:2) nitrate transporter (NRT2.5) polypeptide.
  • Ceres cDNA ID 1828694 (SEQ ID NO:21) is a genomic DNA clone that is predicted to encode a 514 amino acid (SEQ ID NO:22) ammonium transporter (AMTl ;2) polypeptide.
  • Ceres cDNA ID 3086062 (SEQ TD NO:35) is a cDNA clone that is predicted to encode a 557 amino acid (SEQ ID NO: 36) putative transporter polypeptide.
  • Ceres cDNA ID 3091277 (SEQ ID NO: 39) is a cDNA clone that is predicted to encode a 589 amino acid (SEQ ID NO:40) proton-dependent oligopeptide transport (POT) family polypeptide.
  • Ceres cDNA ID 2997404 (SEQ ID NO: 54) is a genomic DNA clone that is predicted to encode a 466 amino acid (SEQ ID NO:55) putative transporter polypeptide.
  • Ceres cDNA ID 4904707 is a cDNA clone that is predicted to encode a 442 amino acid (SEQ ID NO:67) proline transporter 1 (ProTl) polypeptide.
  • Ceres cDNA ID 5669462 is a genomic DNA clone that is predicted to encode a 276 amino acid (SEQ ID NO:83) anthranilate synthase beta chain polypeptide.
  • NB42-35S binary vectors were constructed that contained Ceres cDNA ID 3080447, Ceres cDNA ID 1828694, Ceres cDNA ID 3086062, Ceres cDNA ID 3091277, or Ceres cDNA ID 2997404 operably linked to the cauliflower mosaic virus (CaMV) 35S regulatory region.
  • the NB42-35S binary vector is a derivative of the pMOG800 binary vector.
  • a Ti plasmid vector, CRS 338 was constructed that contained Ceres cDNA ID 4904707 operably linked to the CaMV 35S regulatory region.
  • Another CRS 338 vector was constructed containing Ceres cDNA ID 5669462 operably linked to a regulatory region set forth in SEQ ID NO:93. Wild-type Arabidopsis thaliana ecotype C24 plants were transformed separately with each NB42-35S binary vector containing Ceres cDNA ID 3080447, Ceres cDNA ID 1828694, Ceres cDNA K) 3086062, Ceres cDNA ID 3091277, or Ceres cDNA ID 2997404.
  • Wild-type Arabidopsis thaliana ecotype Ws plants were transformed separately with each Ti plasmid vector containing Ceres cDNA ID 4904707 or Ceres cDNA ID 5669462. The transformations were performed essentially as described in Bechtold et ah, CR. Acad. Sci. Paris, 316:1194-1199 (1993).
  • Transgenic Arabidopsis lines containing Ceres cDNA K) 3080447, Ceres cDNA ID 1828694, Ceres cDNA ID 3086062, Ceres cDNA ID 3091277, Ceres cDNA TD 2997404, Ceres cDNA TD 4904707, or Ceres cDNA ID 5669462 were designated SR00882, SR05002, SR05003, SR05004, SR05005, ME06182, or ME08125, respectively.
  • Arabidopsis ecotype C24 plants were transformed with the empty vector NB42- 35S. As controls for transgenic Arabidopsis ecotype Ws plants, wild-type Arabidopsis ecotype Ws plants were transformed with the empty vector CRS 338.
  • the inplanta nucleotide sequences of Ceres cDNA ID 3080447, Ceres cDNA K) 1828694, Ceres cDNA K) 3086062, Ceres cDNA K) 3091277, Ceres cDNA K) 2997404, Ceres cDNA K) 4904707, and Ceres cDNA ID 5669462 were compared to the homologous Arabidopsis ecotype Columbia sequences.
  • the inplanta sequence of Ceres cDNA K) 1828694 differed from the homologous Columbia sequence by six single nucleotide polymorphisms (SNPs) that resulted in two amino acid changes.
  • the inplanta sequence of Ceres cDNA TD 3086062 differed from the homologous Columbia sequence by 20 SNPs that resulted in nine amino acid changes.
  • the in planta sequences of Ceres cDNA ID 3080447, Ceres cDNA TD 3091277, and Ceres cDNA K) 5669462 matched the homologous Columbia sequences.
  • the inplanta sequence of Ceres cDNA ID 2997404 differed from the homologous Columbia sequence in that it contained a nucleotide insertion near the 3' end, which resulted in a frameshift and a premature stop codon.
  • the in planta nucleotide sequence of Ceres cDNA ID 4904707 differed from the Columbia sequence by four SNPs that did not result in any amino acid changes.
  • Transgenic Arabidopsis lines were screened as follows: 1) T] candidates in the greenhouse were screened for morphological phenotypes, 2) T 2 seeds were analyzed for carbon and nitrogen content, 3) increased carbon and/or nitrogen content was confirmed in T 3 seeds, and 4) T 2 plants were evaluated for negative phenotypes and FinaleTM segregation.
  • Example 2 Analysis of carbon and nitrogen content in transgenic Arabidopsis seeds
  • transgenic Arabidopsis seeds Approximately 2.00 ⁇ 0.15 mg of dried transgenic Arabidopsis seeds (about 100 seeds) were weighed into a tin cup and analyzed for total carbon and nitrogen content. Three matched controls were prepared in a manner identical to the experimental samples and spaced evenly throughout the batch. The first three samples in every batch were a blank (empty tin cup), bypass, (approximately 5 mg of aspartic acid), and a standard (5.00 ⁇ 0.15 mg aspartic acid), respectively. Aspartic acid was weighed into a tin cup using an analytical balance. Blanks were entered between every 15 experimental samples.
  • Thermo Finnigan San Jose, CA.
  • the instrument parameters were as follows: left furnace 900 0 C, right furnace 84O 0 C, oven 5O 0 C, gas flow carrier 130 mL/min., and gas flow reference 100 mL/min.
  • the data parameter LLOD was 0.25 mg for the standard and different for other materials.
  • the data parameter LLOQ was 3 mg for the standard, 1 mg for seed tissue, and different for other materials.
  • Instrument maintenance and performance management included removal of ashes after every 85 analyses, change of oxidation catalyst in the left (reaction) chamber after every 1000 analyses, change of copper in the right (copper reduction) chamber after every 375 analyses, and change of Mg(C10 4 ) 2 after every 250 analyses, or more often if the samples had moisture in them.
  • Quantification was performed using EA 1112 software. The results were normalized and expressed in absolute percentages. Each sample was analyzed in triplicate, and the standard deviation was calculated. Non-transgenic controls were previously determined to have a total carbon content of 53.3 ⁇ 2.4% and a total nitrogen content of 3.9 ⁇ 0.3%. The deviation from theoretical of the aspartic acid standard was ⁇ 2.0% for carbon and ⁇ 1.0% for nitrogen. To be declared valid, each run. was required to have an aspartic acid (standard) weight of 5 mg ⁇ 0.15 mg, and the blank(s) were required to have no recorded nitrogen or carbon content. The percent standard deviation between replicate samples was required to be below 10%.
  • the carbon content of T 2 seeds from two events of SR00882 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 1, the carbon content was increased to 112% in seeds from events -02 and -03 compared to the carbon content in control seeds.
  • Table 1 Total carbon content (% control) of T 2 and T 3 seeds from SR00882 events
  • the carbon content of T 3 seeds from two events of SR00882 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 1, the carbon content was increased to 117% and 112% in seeds from events -02 and -03, respectively, compared to the carbon content in control seeds.
  • T 2 and T 3 seeds from SR00882 events were not observed to differ significantly from the nitrogen content of corresponding control seeds.
  • T 3 seeds from SR00882 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • T 2 SR00882 There were no observable or statistically significant differences between T 2 SR00882 and control plants in germination, onset of flowering, rosette area, fertility, plant height, and general morphology/architecture.
  • Example 4 Results for SR05002 events T 2 and T 3 seeds from three events of SR05002 containing Ceres cDNA
  • the carbon content of T 2 seeds from three events of SR05002 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 2, the carbon content was increased to 108%,
  • Table 2 Total carbon content (% control) of T 2 and T 3 seeds from SR05002 events
  • the nitrogen content of T 2 seeds from three events of SR05002 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 3, the nitrogen content was decreased to 85%, 90%, and 86% in seeds from events -01, -03, and -06, respectively, compared to the nitrogen content in control seeds.
  • Table 3 Total nitrogen content (% control) of T 2 and T 3 seeds from SR05002 events
  • the carbon content of T 3 seeds from three events of SR05002 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 2, the carbon content was increased to 107%, 111%, and 109% in seeds from events -01, -03, and -06, respectively, compared to the carbon content in control seeds.
  • the nitrogen content of T 3 seeds from two events of SR05002 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 3, the nitrogen content was decreased to 87% and 91% in seeds from events -01 and -06, respectively, compared to the nitrogen content in control seeds.
  • T 3 seeds from SR05002 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • T 2 SR05002 There were no observable or statistically significant differences between T 2 SR05002 and control plants in germination, onset of flowering, rosette area, fertility, seed size, and general morphology/architecture.
  • T 2 and T 3 seeds from two events of SR05003 containing Ceres cDNA ID
  • the carbon content of T 2 seeds from two events of SR05003 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 4, the carbon content was increased to 107% and 103% in seeds from events -01 and -04, respectively, compared to the carbon content in control seeds.
  • Table 4 Total carbon content (% control) of T 2 and T 3 seeds from SR05003 events
  • the nitrogen content of T 2 seeds from two events of SR05003 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 5, the nitrogen content was decreased to 89% in seeds from events -01 and -04 compared to the nitrogen content in control seeds.
  • Table 5 Total nitrogen content (% control) of T 2 and T 3 seeds from SR05003 events
  • the carbon content of T 3 seeds from two events of SR05003 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 4, the carbon content was increased to 108% and 107% in seeds from events -01 and -04, respectively, compared to the carbon content in control seeds.
  • the nitrogen content of T 3 seeds from two events of SR05003 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 5, the nitrogen content was decreased to 81% and 92% in seeds from events -01 and -04, respectively, compared to the nitrogen content in control seeds.
  • T 3 seeds from SR05003 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • T 2 SR05003 There were no observable or statistically significant differences between T 2 SR05003 and control plants in germination, onset of flowering, rosette area, fertility, seed size, and general morphology/architecture.
  • the carbon content of T 2 seeds from two events of SR05004 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 6, the carbon content was increased to 122% and 115% in seeds from events -01 and -02, respectively, compared to the carbon content in control seeds.
  • Table 6 Total carbon content (% control) of T 2 and T 3 seeds from SR05004 events
  • T 2 seeds from one event of SR05004 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 7, the nitrogen content was decreased to 74% in seeds from event -02 compared to the nitrogen content in control seeds.
  • Table 7 Total nitrogen content (% control) of T 2 and T 3 seeds from SR05004 events
  • the carbon content of T 3 seeds from two events of SR05004 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 6, the carbon content was increased to 107% and 108% in seeds from events -01 and -02, respectively, compared to the carbon content in control seeds.
  • T 3 seeds from one event of SR05004 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 7, the nitrogen content was decreased to 87% in seeds from event -02 compared to the nitrogen content in control seeds.
  • T 3 seeds from SR05004 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event. The segregation of FinaleTM resistance in T 2 plants from events -01 and -
  • 02 of SR05004 was a 3:1 ratio of resistant to sensitive.
  • T 2 SR05004 There were no observable or statistically significant differences between T 2 SR05004 and control plants in germination, onset of flowering, rosette area, fertility, seed size, and general morphology/architecture.
  • T 2 and T 3 seeds from two events of SR05005 containing Ceres cDNA ID 2997404 were analyzed for total carbon and nitrogen content as described in Example 2.
  • the carbon content of T 2 seeds from two events of SR05005 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 8, the carbon content was increased to 114% and 105% in seeds from events -01 and -04, respectively, compared to the carbon content in control seeds.
  • Table 8 Total carbon content (% control) of T 2 and T 3 seeds from SR05005 events
  • the nitrogen content of T 2 seeds from two events of SR05005 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 9, the nitrogen content was decreased to 85% and 84% in seeds from events -01 and -04, respectively, compared to the nitrogen content in control seeds.
  • Table 9 Total nitrogen content (% control) of T 2 and T 3 seeds from SR05005 events
  • the carbon content of T 3 seeds from two events of SR05005 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 8, the carbon content was increased to 104% and 105% in seeds from events -01 and -04, respectively, compared to the carbon content in control seeds.
  • the nitrogen content of T 3 seeds from one event of SR05005 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 9, the nitrogen content was decreased to 92% in seeds from event -01 compared to the nitrogen content in control seeds.
  • T 3 seeds from SR05005 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • the segregation ratio of FinaleTM resistance in T 2 plants from events -01 and -04 of SR05005 was 3:1 resistant to sensitive.
  • T 2 SR05005 There were no observable or statistically significant differences between T 2 SR05005 and control plants in germination, onset of flowering, rosette area, fertility, seed size, and general morphology/architecture.
  • the carbon content of T 2 seeds from two events of ME06182 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 10, the carbon content was increased to 104% in seeds from events -02 and -03 compared to the carbon content in control seeds.
  • Table 10 Total carbon content (% control) of T 2 and T 3 seeds from ME06182 events
  • the nitrogen content of T 2 seeds from two events of ME06182 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 11, the nitrogen content was decreased to 77% and 87% in seeds from events -02 and -03, respectively, compared to the nitrogen content in control seeds.
  • Table 11 Total nitrogen content (% control) of T 2 and T 3 seeds from ME06182 events
  • the carbon content of T 3 seeds from two events of ME06182 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 10, the carbon content was increased to 105% and 106% in seeds from events -02 and -03, respectively, compared to the carbon content in control seeds.
  • the nitrogen content of T 3 seeds from one event of ME06182 was significantly decreased compared to the nitrogen content of corresponding control seeds. As presented in Table 11, the nitrogen content was decreased to 94% in seeds from event -02 compared to the nitrogen content in control seeds.
  • T 3 seeds from ME06182 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • Example 9 Results for ME08125 events T 2 and T 3 seeds from two events of ME08125 containing Ceres cDNA ID
  • the carbon content of T 2 seeds from two events of ME08125 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 12, the carbon content was increased to 104% in seeds from events -03 and -04 compared to the carbon content in control seeds.
  • Table 12 Total carbon content (% control) of T 2 and T 3 seeds from ME08125 events
  • the nitrogen content of T 2 seeds from two events of ME08125 was significantly increased compared to the nitrogen content of corresponding control seeds. As presented in Table 13, the nitrogen content was increased to 116% and 112% in seeds from events -03 and -04, respectively, compared to the nitrogen content in control seeds.
  • Table 13 Total nitrogen content (% control) of T 2 and T 3 seeds from ME08125 events
  • the carbon content of T 3 seeds from two events of ME08125 was significantly increased compared to the carbon content of corresponding control seeds. As presented in Table 12, the carbon content was increased to 105% and 103% in seeds from events -03 and -04, respectively, compared to the carbon content in control seeds.
  • T 3 seeds from ME08125 events analyzed for carbon and nitrogen content were collected from one T 2 plant from each event.
  • Example 10 Determination of functional homolog and/or ortholos sequences A subject sequence was considered a functional homolog or ortholog of a query sequence if the subject and query sequences encoded proteins having a similar function and/or activity. A process known as Reciprocal BLAST (Rivera et al, Proc. Natl. Acad.
  • the main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search.
  • forward search step a query polypeptide sequence, "polypeptide A,” from source species SA was BLASTed against all protein sequences from a species of interest. Top hits were determined using an E-value cutoff of 10-5 and an identity cutoff of 35%.
  • top hits the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
  • top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA.
  • a top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog or ortholog.
  • Functional homologs and/or orthologs were identified by manual inspection of potential functional homolog and/or ortholog sequences.
  • Representative functional homologs and/or orthologs for SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:83 are shown in Figures 1-7, respectively.
  • the percent identities of functional homologs and/or orthologs to SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:67, and SEQ ID NO:83 are shown below in Tables 14-20, respectively.
  • Table 14 Percent identity to Ceres cDNA ID 3080447 (SEQ ID NO:2)
  • Table 17 Percent identity to Ceres cDNA ID 3091277 (SEQ ID NO:40)
  • Table 18 Percent identity to Ceres cDNA ID 2997404 (SEQ ID NO:55)

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Abstract

L'invention concerne des procédés et des matériaux de modulation (par exemple, augmentation ou diminution) des teneurs en carbone dans les plantes. A titre d'exemple, l'invention concerne des acides nucléiques codant des polypeptides modulant le carbone, ainsi que des procédés d'utilisation de tels acides nucléiques en vue de transformer des cellules végétales. L'invention concerne en outre des plantes présentant des teneurs en carbone accrues.
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WO2013140155A1 (fr) * 2012-03-20 2013-09-26 British Americal Tobacco (Investments) Limited Plantes transgéniques ayant des taux de nitrate modifiés dans les feuilles
CN104395473A (zh) * 2012-03-20 2015-03-04 英美烟草(投资)有限公司 具有叶中改变的硝酸盐水平的转基因植物
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US10597666B2 (en) 2012-03-20 2020-03-24 British American Tobacco (Investments) Limited Transgenic plants with altered nitrate levels in leaves
CN108866082A (zh) * 2018-08-06 2018-11-23 南京农业大学 大豆STF-3转录因子编码基因GmSTF-3及其应用
CN113234735A (zh) * 2021-06-07 2021-08-10 山西省林业和草原科学研究院 杨树PtNF-YC1基因及其在促进植物提前开花中的应用
CN113234735B (zh) * 2021-06-07 2022-12-23 山西省林业和草原科学研究院 杨树PtNF-YC1基因及其在促进植物提前开花中的应用

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